Gram-negative sepsis remains a major cause of mortality and morbidity in hospitalized patients, especially when complicated by acute kidney injury (AKI). The pathophysiology of AKI in sepsis continues to be poorly understood resulting in the persistent failure of clinical therapeutic trial. Recently, we identified a novel pathway of renal injury in sepsis involving direct interactions between filtered endotoxin and S1 proximal tubules. This endotoxin-S1 interaction resulted in severe peroxisomal damage and oxidative stress in downstream S2 and S3 segments. Remarkably, this pathway of injury had no requirement for competent immune cells. In this proposal, we continue our investigation of sepsis and AKI by examining the mechanisms of renal endotoxin preconditioning. The phenomenon of protective preconditioning is unique in that it represents a state of resistance to the deleterious effects of endotoxin and yet, a preserved ability to effectively contain and eliminate infections. Unraveling the pathways involved in endotoxin tolerance has great potential in identifying potential targets that can be used for the prevention and treatment of human sepsis. Historically, preconditioning has been investigated in immune cells and their isolated responses to repeated endotoxin exposure. Little is known about the mechanisms leading to tissue protection in whole organs such as the kidney. Based on strong preliminary data, the central hypothesis of this proposal is that macrophages are essential components of the protective pathways of preconditioning. This is a novel hypothesis because it depicts the macrophage as an active and beneficial participant in the tolerant phenotype. In specific aim 1, we will establish the essential role of macrophages in inducing renal endotoxin tolerance by examining chimeric mice lacking myeloid TLR4 and CD14. We will fully characterize these macrophages by flow techniques and image their spatial and temporal interactions with renal tubules using intra vital microscopy. In specific aim 2, we will examine th potential of protective macrophages to prevent and treat sepsis using cell transfer approaches. In specific aim 3, we will examine in detail the roles of macrophage CD14, heme oxygenase 1 (HO1) as essential molecules of the macrophage phenotype necessary for inducing renal protection. We will also examine the potential roles of various metabolites involved in macrophage differentiation and function as therapeutic tools against sepsis. These three aims will identify the macrophage as the most central player in renal protective preconditioning. These aims will also identify pathways controlled by CD14 and TRIF that are potential targets for therapeutic intervention in septic patients. We believe that the proposed studies, by increasing our understanding of endotoxin preconditioning, have great translational potential and will uncover a novel and global approach to the prevention and treatment of sepsis and sepsis-induced AKI.